The goal of this research is to obtain a better mechanistic understanding of the factors underlying the functional specificity of Drosophila homeotic and mammalian Hox proteins, particularly those in the Deformed-like class. The core DNA sequence elements that are sufficient to confer Deformed specificity on a regulatory element in the context of an embryo are unknown. This proposal aims at the definition of such elements through mutagenesis studies on two Deformed autoregulatory enhancers (one specific for epidermal cells, and one for central nervous system cells). In addition, a human autoregulatory element that supplies maxillary segment-specific expression in Drosophila will be dissected to test whether it also contains Deformed response elements similar to those present in Drosophila enhancers. Using test of the function of Deformed/Ultrabithorax hybrid protein expression in Drosophila under heat shock promoter control, the targeting specificity of the Deformed and Ultrabithorax proteins will be analyzed. Affinity methods will be used to screen for proteins that interact with Deformed homeodomain proteins. As another approach to identify proteins that interact with, and contribute to the regulatory specificity of the Deformed protein, we will screen for mutations that enhance Deformed mutant phenotypes. Expression of some mouse and human Hox proteins in Drosophila causes gain-of-function mutations similar to the phenotypes caused by their Drosophila homologs. One simple hypothesis to explain this is that the mouse and human Hox proteins may conserve cross-regulatory relationships with extended members of the HOM/Hox system that may include the ems, Dll, otd, lab, and tsh genes. We will test this hypothesis using genetic epistasis tests and analysis of expression patterns in Hox protein expressing Drosophila embryos.